Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D307/87—Benzo [c] furans; Hydrogenated benzo [c] furans
  • C07D307/88—Benzo [c] furans; Hydrogenated benzo [c] furans with one oxygen atom directly attached in position 1 or 3
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
  • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/465—Streptomyces
  • C12R2001/485—Streptomyces aureofaciens
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/465—Streptomyces
  • C12R2001/51—Streptomyces candidus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/886—Streptomyces
  • Y10S435/892—Streptomyces candidus

Definitions

• This invention relates to processes for preparing mycophenolic acid glucoside which is useful in the treatment of psoriasis and gout. It also inhibits the growth of transplanted tumor cells in mice and rats.
• Mycophenolic acid and mycophenolic acid glucoside have structures 1 and 2, respectively:
• a process for preparing mycophenolic acid glucoside which comprises contacting mycophenolic acid with glucose in the presence of a glucosylating enzyme in an aqueous medium until a substantial amount of mycophenolic acid glucoside is produced.
• glucosylating enzyme useful in the process of the invention there may be mentioned the enzyrneproduced by Streptomyces candidus NRRL 5449 and that produced by Streptomyces aureofaciens NRRL 2209.
• either purified mycophenolic acid or partially purified mycophenolic acid may be used as a starting material.
• the preferred enzyme which glucosylates mycophenolic acid is produced by a strain of S. aureofaciens NRRL 2209 or S. candidus NRRL 5449. The latter is also the subject of our U.S. Patent 3,932,619.
• the culture medium used to grow S. aureofaciens NRRL 2209 or S. candidus NRRL 5449 can be any one of a number of media. For optimal transformation, however, certain culture media are preferred. These media should contain assimilable sources of carbon, nitrogen, and inorganic salts. Preferred sources of carbon are reducing sugars; and a preferred source of nitrogen is soybean meal.
• the medium When the conversion is to take place in the fermentation culture at the the time S. aureofaciens NRRL 2209 or S. candidus NRRL 5449 is grown, the medium must contain a source of glucose to achieve efficient conversion. When the microorganism is grown to produce its enzyme for later use in converting mycophenolic acid to mycophenolic acid glucoside, the presence of glucose during fermentation is optional.
• Essential trace elements necessary for the growth and development.of the organism may occur as impurities in other constituents of the media in amounts sufficient to meet the growth and biosynthetic requirements of the organism. It may be beneficial, however, to incorporate in the culture media additional soluble nutrient inorganic salts capable of yielding iron, sodium, potassium, magnesium, ammonium, calcium, phosphate, chloride, carbonate, sulfate, nitrate and like ions.
• the initial pH of the culture medium can be varied. Prior to inoculation with the organism, however, it is desirable to adjust the pH of the culture medium to between 5.7 and 7.5, depending upon the particular medium used. As is the case with other Actinomycetes, the medium gradually becomes more alkaline as the fermentation proceeds and may rise from an initial pH of 5.9 to 6.9 or higher during the growth period of the organism.
• the final pH is influenced by the initial pH of the medium, the substrate and buffers present in the medium, and the duration of time the organism is permitted to grow. Although pH can be adjusted by addition of either acid or base, good results have been achieved with no adjustment of pH.
• the microorganisms require aerobic growth conditions. Small-volume propagation is conveniently carried out on agar slants or plates, in shake flasks or in bottles. For large-scale production, submerged aerobic culture in large tanks is preferred.
• the fermentation medium in a sterile tank can be inoculated with a sporulated suspension to initiate fermentation. Since inoculation with a sporulated suspension involves a growth lag, however, a vegetative inoculum is preferable.
• the vegetative inoculum can be prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank.
• the medium used for the growth of the vegetative inoculum can be the same as that used for large-scale production, but other media can also be used.
• the organism S. aureofaciens NRRL 2209 will grow over a temperature range between 20°C. to 37°C. Maximum growth and sporulation, however, occur between 28°C. and 32°C.
• the S. candidus NRRL 5449 organism will grow over a temperature range between 26°C. to 40°C., with maximum growth and sporulation occuring between 32°C. and 37°C.
• the volume of air used in tank production of the substance should be above -0.1 volume of. air per volume of culture medium per minute (V/V/M). Optimal yields are obtained when the volume of air used is at least one-third to one-half volume of air per volume of culture medium per minute.
• the fermentation time needed to effect the conversion varies.
• the presence of an adequate supply of glucose is essential.
• mycophenolic acid is present in an amount of 0.1 to 1.0 grams per liter of medium
• conversion of mycophenolic acid to mycophenolic acid glucoside is essentially complete in from 72 to 120 hours.
• Optimal conversion occurs when mycophenolic acid is present in a range of from 0.2 to 0.75 grams per liter of medium.
• An adequate amount of glucose is above one percent of medium by weight.
• a preferred amount of glucose is from two to five percent of medium by weight.
• a glucose-sensing test paper may be used to check concentration levels. When glucose content drops below about two percent, glucose should be added to maintain concentration at optimum levels.
• Yet another method of carrying out the conversion is to immobilize the enzyme by methods known in the art. (See, for example, “Biomedical Applications of Immobilized Enzymes and Proteins", Thomas Ming Swi Chang, Ed., Plenum Press, New York, 1977; Vol. l.)
• the immobilized enzyme can then be used in a column (or other suitable type of reactor) to effect the glucosylation reaction.
• microorganism itself can be immobilized and used to catalyze the glucosylation reaction.
• TLC thin-layer chromatography
• Mycophenolic acid glucoside is present in both the culture broth and in the mycelia. Accordingly, techniques used in the isolation of mycophenolic acid glucoside should be designed to permit maximum recovery of the product from either or both sources.
• the fermentation medium is filtered, the pH of the filtrate is lowered to the acidic range, and the acidified filtrate is extracted with a suitable solvent such as chloroform to remove any unreacted mycophenolic acid.
• the mycelial cake is extracted with a suitable solvent such as methanol; the organic solvent is removed; and the aqueous solution remaining is added to the filtered broth for the isolation steps.
• the product is recovered from the solution by methods known in the art. A preferred method of recovery is by adsorption over polymeric resin and elution with a suitable solvent system, such as methanolwater.
• a lyophilized pellet of Streptomyces aureofaciens NRRL 2209 was suspended in water and used to inoculate an agar slant having the following composition: FeSO 4 ⁇ 7H 2 O (2 g) was dissolved in 100 ml deionized water containing 2 ml of concentrated HC1. This solution was added to the above KCl/MgSO 4 ⁇ 7H 2 O solution to complete preparation of the Czapek's Minerals.
• Inoculated slants were incubated at 30°C for 10 days and then stored at 4°C for no more than 30 days.
• a loop inoculum from a slant culture was transferred into.50 ml of vegetative medium in a 250-ml Erlenmeyer flask.
• the vegetative medium had the following composition:
• the inoculated flask was incubated at 30°C on a rotary shaker operating at 250 rpm. After 48 hours of incubation, 5 ml of culture were transferred to 100 ml of second-stage vegetative medium (having the same composition as the vegetative medium) in a 500-ml Erlenmeyer flask. This culture was incubated for 48 hours at 30°C on a rotary shaker operated at 250 rpm.
• Mycophenolic acid (50 mg) was dissolved in water (8 ml). The pH was adjusted to 7.0, and water was added to a volume of 10 ml. This solution was added to the flask obtained in Sect. A which contained the S. aureofaciens enzyme along with 4 ml of an aqueous solution containing 250 mg/ml of glucose. The flask was then incubated an additional 72 hours.
• S. aureofaciens NRRL 2209 is grown on an agar slant prepared from Bennett's medium. The growth is removed and made up as a slurry with sterile deionized water (10 ml).
• This slurry is divided among four 500-ml shake flasks, each containing 100 ml of defined vegetative medium of the following composition:
• the four inoculated flasks are incubated at 30°C. on a rotary shaker at 250 rpm for 48 hours.
• This vegetative medium (5-ml portions) is used to inoculate shake flasks (500 ml) containing 100 ml of defined fermentation medium having the same composition as the defined vegetative medium.
• the inoculated medium is incubated for 48 hours.
• Mycophenolic acid glucoside is prepared by the method described in Example 1 except that the enzyme is that produced by Streptomyces candidus NRRL 5449 by alternate step A as follows:
• S. candidus NRRL 5449 is grown on an agar slant prepared from Bennett's medium to give a well- defined colony. The colony is removed and made up as a slurry with sterile deionized water (10 ml).
• This slurry is divided among four 500-mi shake flasks, each containing 100 ml of vegetative medium of the following composition:
• the four inoculated flasks are incubated at 30°C. on a rotary shaker at 250 rpm for 24 hours.
• This vegetative medium (10-ml portions) is used to inoculate shake flasks (500 ml) containing 100 ml of sterilized fermentation medium of the following composition:
• the inoculated medium is incubated for 72 hours as described above.
• S. candidus NRRL 5449 is grown as described in Example 3.
• the cells are separated from the fermentation medium by vacuum filtration and are divided into 200-g aliquots which are stored by freezing. Two of these aliquots are thawed at room temperature and are suspended in 0.05 M phosphate buffer (pH 5.8) to a final volume of 600 ml. This cell suspension is sonicated for 30 minutes, and the sonicate is centrifuged at 10,000 rpm for 30 minutes. The resulting cell debris is suspended in 100 ml of the phosphate buffer; this suspension is dialyzed for 18 hours with 5 L of the chilled, phosphate buffer.
• LP-1 silica gel 1000 g from Quantum Corp., now Whatman is added to a mixture of concentrated sulfuric acid (1650 ml) and concentrated nitric acid (1650 ml) in a 5-L round-bottom flask and shaken for proper suspension. The mixture is heated on a steam bath overnight (16 hours) with a water-jacketed condenser attached to the flask.
• the mixture is cooled in an ice bath and carefully filtered using a sintered-glass funnel.
• the silica gel is washed with deionized water until the pH is neutral.
• the silica gel is then washed with acetone (4 L) and dried under vacuum at 100°C. for 2 days.
• the dry silica gel from Step 1 is transferred to a round-bottom flask and suspended in toluene (3.5 L). The flask is heated on a steam bath for 2 hours to azeotrope off some residual water. Octadecyltrichlorosilane (321 ml, Aldrich Chemical Company) is added, and the reaction mixture is refluxed overnight (16 hours) with slow mechanical stirring at about 60°C. Care is taken so that the stirrer does not reach near the bottom of the flask. This is to prevent grinding the silica gel particles.
• silan- ized silica gel is collected, washed with toluene (3 L) and acetone (3 L), and then air-dried overnight (16-20 hours).
• the dried silica gel is suspended in 3.5 L of acetonitrile:water (1:1) in a 5-L flask, stirred carefully at room temperature for 2 hours, filtered, washed with acetone (3 L) and air-dried overnight.
• Analytical or preparative columns can be packed by this procedure.
• silica gel reversed phase packings e.g., Quantum LP-1, particle size 10-20 microns; LiChroprep RP-8 and RP-18, particle size 25-40 microns
• silica gels e.g., Shandons ODS Hypersil, particle size 5 microns
• other types of resins e.g., Shandons ODS Hypersil, particle size 5 microns
• the time required to pack a glass column will vary from minutes to several hours depending on column size and experience of the scientist.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Virology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Medicinal Chemistry (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Saccharide Compounds (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=22290205

Family Applications (1)

Country Status (12)

Families Citing this family (7)

Family Cites Families (2)

• 1979
  • 1979-12-11 US US06/102,504 patent/US4234684A/en not_active Expired - Lifetime
• 1980
  • 1980-11-03 AR AR283106A patent/AR224657A1/es active
  • 1980-12-02 BE BE1/10057A patent/BE886438A/fr not_active IP Right Cessation
  • 1980-12-02 FR FR8025581A patent/FR2471409A1/fr not_active Withdrawn
  • 1980-12-03 IL IL61617A patent/IL61617A0/xx unknown
  • 1980-12-04 EP EP80304387A patent/EP0030460A1/de not_active Ceased
  • 1980-12-04 GB GB8038911A patent/GB2065119B/en not_active Expired
  • 1980-12-05 IE IE2542/80A patent/IE50522B1/en unknown
  • 1980-12-09 IT IT26523/80A patent/IT1134642B/it active
  • 1980-12-10 JP JP17523280A patent/JPS56121493A/ja active Pending
  • 1980-12-10 HU HU802962A patent/HU182255B/hu unknown
  • 1980-12-11 KR KR1019800004703A patent/KR840000892B1/ko active

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events